Berberine bridge enzyme-like oxidase-catalysed double bond isomerization acts as the pathway switch in cytochalasin synthesis

Cytochalasans (CYTs), as well as their polycyclic (pcCYTs) and polymerized (meCYTs) derivatives, constitute one of the largest families of fungal polyketide-nonribosomal peptide (PK-NRP) hybrid natural products. However, the mechanism of chemical conversion from mono-CYTs (moCYTs) to both pcCYTs and meCYTs remains unknown. Here, we show the first successful example of the reconstitution of the CYT core backbone as well as the whole pathway in a heterologous host. Importantly, we also describe the berberine bridge enzyme (BBE)-like oxidase AspoA, which uses Glu538 as a general acid biocatalyst to catalyse an unusual protonation-driven double bond isomerization reaction and acts as a switch to alter the native (for moCYTs) and nonenzymatic (for pcCYTs and meCYTs) pathways to synthesize aspochalasin family compounds. Our results present an unprecedented function of BBE-like enzymes and highly suggest that the isolated pcCYTs and meCYTs are most likely artificially derived products.

Glycosidically-Bound Volatile Phenols Linked to Smoke Taint: Stability during Fermentation with Different Yeasts and in Finished Wine

When wine grapes are exposed to smoke, there is a risk that the resulting wines may possess smoky, ashy, or burnt aromas, a wine flaw known as smoke taint. Smoke taint occurs when the volatile phenols (VPs) largely responsible for the aroma of smoke are transformed in grape into a range of glycosides that are imperceptible by smell. The majority of VP-glycosides described to date are disaccharides possessing a reducing β-d-glucopyranosyl moiety. Here, a two-part experiment was performed to (1) assess the stability of 11 synthesized VP-glycosides towards general acid-catalyzed hydrolysis during aging, and (2) to examine whether yeast strains differed in their capacity to produce free VPs both from these model glycosides as well as from grapes that had been deliberately exposed to smoke. When fortified into both model and real wine matrices at 200 ng/g, all VP-disaccharides were stable over 12 weeks, while (42–50 ng/g) increases in free 4-ethylphenol and p-cresol were detected when these were added to wine as their monoglucosides. Guaiacol and phenol were the most abundantly produced VPs during fermentation, whether originating from natural VP-precursors in smoked-exposed Pinot Noir must, or due to fortification with synthetic VP-glycosides. Significant yeast strain-specific differences in glycolytic activities were observed for phenyl-β-d-glycopyranoside, with two strains (RC212 and BM45) being unable to hydrolyze this model VP, albeit both were active on the guaiacyl analogue. Thus, differences in Saccharomyces cerevisiae β-glucosidase activity appear to be influenced by the VP moiety.

Identification of general acid catalyst for the ATPase activity of Lynch syndrome-related MutL homologs

Mutations of mismatch repair MutL homologs are causative of a hereditary cancer, Lynch syndrome. Investigation of MutL facilitates genetic diagnoses essential for cancer risk managements and therapies. We characterized MutL homologs from human and a hyperthermophile, Aquifex aeolicus, (aqMutL) to reveal the catalytic mechanism for the ATPase activity. Although existence of a general acid catalyst had not been conceived in the mechanism, analysis of the pH dependence of the aqMutL ATPase activity revealed that the reaction is accelerated by general acid-base catalysis. Analyses of mutant aqMutLs showed that Lys79 is the general acid, and the corresponding residues were confirmed to be critical for activities of human homologs, on the basis of which a catalytic mechanism for MutL ATPase is proposed. These and other results described here would contribute to evaluating the pathogenicity of Lynch syndrome-associated missense mutations.

Crystal Structures of Streptomyces Coelicolor Methylmalonyl-CoA Epimerase with Substrate or Transition State Analog Contradicts a Simple General Acid-Base Catalytic Mechanism

Crystal structures of Streptomyces coelicolor methylmalonyl-CoA epimerase in the holo-form, with substrate or the putative transition state analog, 2-nitroproionyl-CoA. The proposed catalytic mechanism is general acid-base catalysis. The proposed catalytic residues are too far from the substrate or analog, unless conformational changes take place or some other mechanism is used. <br>

Crystal Structures of Streptomyces Coelicolor Methylmalonyl-CoA Epimerase with Substrate or Transition State Analog Contradicts a Simple General Acid-Base Catalytic Mechanism

Crystal structures of Streptomyces coelicolor methylmalonyl-CoA epimerase in the holo-form, with substrate or the putative transition state analog, 2-nitroproionyl-CoA. The proposed catalytic mechanism is general acid-base catalysis. The proposed catalytic residues are too far from the substrate or analog, unless conformational changes take place or some other mechanism is used. <br>

Gcn5-Related N-Acetyltransferases (GNATs) With a Catalytic Serine Residue Can Play Ping-Pong Too

Enzymes in the Gcn5-related N-acetyltransferase (GNAT) superfamily are widespread and critically involved in multiple cellular processes ranging from antibiotic resistance to histone modification. While acetyl transfer is the most widely catalyzed reaction, recent studies have revealed that these enzymes are also capable of performing succinylation, condensation, decarboxylation, and methylcarbamoylation reactions. The canonical chemical mechanism attributed to GNATs is a general acid/base mechanism; however, mounting evidence has cast doubt on the applicability of this mechanism to all GNATs. This study shows that the Pseudomonas aeruginosa PA3944 enzyme uses a nucleophilic serine residue and a hybrid ping-pong mechanism for catalysis instead of a general acid/base mechanism. To simplify this enzyme’s kinetic characterization, we synthesized a polymyxin B substrate analog and performed molecular docking experiments. We performed site-directed mutagenesis of key active site residues (S148 and E102) and determined the structure of the E102A mutant. We found that the serine residue is essential for catalysis toward the synthetic substrate analog and polymyxin B, but the glutamate residue is more likely important for substrate recognition or stabilization. Our results challenge the current paradigm of GNAT mechanisms and show that this common enzyme scaffold utilizes different active site residues to accomplish a diversity of catalytic reactions.

Hydrated metal ion as a general acid in the catalytic mechanism of the 8-17 DNAzyme

The RNA-cleaving 8-17 DNAzyme, which is a metalloenzyme that depends on divalent metal ions for function, is the most studied catalytic DNA in terms of its mechanism. By the end...